1. Field of the Invention
This invention relates to methods and materials useful for the detection of melamine.
2. Background.
Melamine (1,3,5-triazine-2,4,6-triamine) is an organic compound used extensively as a raw material in the preparation of polymers for the manufacture of a wide range of products, including fabrics, laminates, adhesives, molding compounds, coatings, and flame retardants. Melamine is also used in some fertilizers and can be found as a metabolite of the pesticide, cyromazine. Despite the widespread use of melamine, consumer exposure to melamine is considered to be low.
Melamine by itself is nontoxic in low doses, but when combined with cyanuric acid it can cause fatal kidney stones due to the formation of an insoluble melamine cyanurate. When melamine and cyanuric acid are absorbed into the bloodstream, they concentrate and interact in the urine-filled renal microtubules, crystallizing and forming large numbers of round, yellow crystals, which in turn block and damage the renal cells that line the tubes, causing the kidneys to malfunction. In low doses melamine is rapidly eliminated in the urine with a half-life in plasma of around three hours. In view of its pharmacokinetic profile, the US FDA has established that the tolerable daily intake (TDI) of melamine for adults is 0.63 mg/kg of body weight.
Melamine contamination in food may be due to migration from industrial uses, metabolic processing of pesticides, i.e. cyromazine, or intentional doping to modify nitrogen content analysis; for example in milk, where it is added to compensate for dilution with water (see, e.g. Szmant, H. H. in Organic Building Blocks of the Chemical Industry 1989, John Wiley and Sons; Hauck et al., Agric. Food Chem. 1964, 12, 147-151; U.S. Pat. No. 2,819,968; Newton et al., R. J. Animal Sci. 1978, 47, 1338-1344; Lim et al., J. Agric. Food Chem. 1990, 38, 860-864). High levels of melamine exposure have been associated with kidney failure and the adulteration of food products with melamine has resulted in the deaths of thousands of people and pets (see, e.g. Puschner et al., J. Vet. Diagn. Inv. 2007, 19, 616-624; Dobson et al., Toxicol. Sci. 2008, 106, 251-262; Hau et al., J. Am. Soc. Nephrol. 2009, 20, 245-250).
In view of its use, for example, as an adulterant of foodstuffs, a number of methods for detecting melamine in test samples have been developed. In conventional methods, a test substance to be analyzed for the presence of melamine is first purified using centrifugation and filtration techniques. Following these purification steps, liquid phase chromatography (LC) or high-pressure liquid chromatography (HPLC) is then performed prior to the final detection of melamine using a mass spectrum/spectrometer. Although these methods and other spectrum-based methods provide accurate techniques in determining melamine content, such methods are very labor intensive, time consuming, and expensive to perform. While enzyme-linked immunosorbent assays (ELISA) are also used to detect melamine, such techniques are similarly labor intensive, time consuming, and expensive to perform.
In view of the above, there is a need for inexpensive, accurate, simple, and fast methods for detecting the presence of melamine in test samples.